Canonical Bone Morphogenetic Protein Signaling Research Articles

Hoxa2 Inhibits Bone Morphogenetic Protein Signaling during Osteogenic Differentiation of the Palatal Mesenchyme.

Cleft palate is one of the most common congenital birth defects worldwide. The homeobox (Hox) family of genes are key regulators of embryogenesis, with Hoxa2 having a direct role in secondary palate development. Hoxa2−/− mice exhibit cleft palate; however, the cellular and molecular mechanisms leading to cleft palate in Hoxa2−/− mice is largely unknown. Addressing this issue, we found that Hoxa2 regulates spatial and temporal programs of osteogenic differentiation in the developing palate by inhibiting bone morphogenetic protein (BMP) signaling dependent osteoblast markers. Expression of osteoblast markers, including Runx2, Sp7, and AlpI were increased in Hoxa2−/− palatal shelves at embryonic day (E) 13.5 and E15.5. Hoxa2−/− mouse embryonic palatal mesenchyme (MEPM) cells exhibited increased bone matrix deposition and mineralization in vitro. Moreover, loss of Hoxa2 resulted in increased osteoprogenitor cell proliferation and osteogenic commitment during early stages of palate development at E13.5. Consistent with upregulation of osteoblast markers, Hoxa2−/− palatal shelves displayed higher expression of canonical BMP signaling in vivo. Blocking BMP signaling in Hoxa2−/− primary MEPM cells using dorsomorphin restored cell proliferation and osteogenic differentiation to wild-type levels. Collectively, these data demonstrate for the first time that Hoxa2 may regulate palate development by inhibiting osteogenic differentiation of palatal mesenchyme via modulating BMP signaling.

Transcription factors-Intricate players of the bone morphogenetic protein signaling pathway.

Bone morphogenetic proteins (BMPs) are a family of growth factors, some of which are known by the name growth and differentiation factor (GDF). BMPs were discovered in the 1960s in an attempt to find factors capable of inducing bone formation. By the end of 1980s, several different BMPs had been found and to date, around 20 members are known. Together with TGFβ, nodal, and activins, they comprise the TGFβ superfamily. BMPs are known to regulate cell fate both in development and adult tissues, and as such they are also involved in many disease states. Understanding the impact of BMPs in these processes requires intimate knowledge of the regulation of the BMP signaling pathway. The intracellular BMP pathway has been studied extensively but the various transcription factors (TFs) necessary for the BMP-mediated gene expression changes have remained under less scrutiny. Most of the studies have focused on specific TFs in the context of differentiation or development. Varying cell types and BMPs have been used, but no large-scale studies have yet been performed. Here we aim to summarize the current knowledge on BMP pathway-related TFs, focusing on those involved in the canonical BMP signaling pathway through the SMAD proteins.

Abstract 3087: Forced induction of differentiation in osteosarcoma tumor initiating cells

Abstract Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years and similarly, the five-year survival rate remains at ~60%, with the majority of fatalities arising from metastases in the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation. In previous work, our lab has developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to green fluorescent protein (GFP) coding sequence. Clonally derived, stably transfected OS Oct4/GFP+ TICs are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment, resulting in adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth. To first probe that OS TICs respond to BMP stimulation, we detected by flow cytometry the expression of BMP Receptors Type II and 1A in two primary OS-derived cell lines (OS521 and OS156), with similar expression seen in Mesenchymal Stem Cells. Furthermore, by treating OS TICs with BMP heterodimers - BMP2/7 and BMP4/7 - we observed an activation of canonical BMP signaling, suggesting a functional and intact signaling network. BMP4/7 stimulation of OS521 TICs in vitro showed an increase in cells in the G1 phase of the cell cycle, visualized by DAPI, and higher p21 and p27 expression than controls, shown in immunoblotting. On the other hand, BMP4/7 treatment of OS156 TICs reduced its invasion capacity through a complex of Fibronectin and Bovine Serum Albumin by 33% (p= 0.00144) compared to untreated controls. Strikingly, in vivo studies revealed that time to tumor onset of BMP4/7 pretreated OS521 TICs in NSG mice at a dose of 3x104 cells was significantly longer than in controls (p=0.0003). In summary, our data suggests a reduction of OS tumorigenicity due to a BMP4/7 induced differentiation-based response. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy. Future studies include TIC treatment with hypoxia, adenosine, and calcium, to investigate the underlying mechanisms responsible for BMP involvement in the tumorigenic capacity of osteosarcoma. Citation Format: Margaret Ellen White, Maria V. Guijarro, Steven Ghivizzani, Charles P. Gibbs. Forced induction of differentiation in osteosarcoma tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2017-3087

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

Abstract 1188: Forced induction of differentiation in osteosarcoma tumor initiating cells

Abstract Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years, and similarly the five-year survival rate has remained a dismal ∼60%, largely due to metastatic spread to the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation. In previous work, our lab developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to the coding sequence for Green Fluorescent Protein (GFP). Clonally derived, stably transfected OS Oct4/GFP+ cells are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment. These conditions appear to reprogram the GFP+ cells, resulting in loss of reporter activity, reduced proliferation, and adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in normal physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth. To test our hypothesis, we investigated the expression of BMP receptors in two primary OS cell lines, using Mesenchymal Stem Cells and Human Fetal Osteoblasts as positive controls. Expression of BMP Receptors II, 1A, and 1B was confirmed using flow cytometry indicating that the OS cells could potentially respond to BMP stimulation. The functionality of the BMP signaling pathway was subsequently investigated after stimulation with BMP heterodimers, BMP2/7 and BMP4/7. Immunoblotting showed OS TICs are capable of activating canonical and non-canonical BMP signaling pathways after BMP stimulation, suggesting a functional and intact signaling network potentially able to induce differentiation. However, only cells treated with BMP4/7 showed changes in cell cycle distribution as visualized by DAPI. Furthermore, BMP4/7-stimulated TICs showed reduced tumor formation in our xenograft mouse model Our data suggests an inhibition of OS tumorigenicity due to a BMP4/7 induced differentiation based response. Currently, we are investigating if forced expression of BMPs using an OS specific recombinant Adeno-Associated Virus can inhibit tumor growth in vivo. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy. Citation Format: Margaret E. White, Emma Viktoria Marie Hyddmark, Ali Zarezadeh, Elham Nasri, Maria V. Guijarro, Padraic P. Levings, Glyn Palmer, Steven C. Ghivizzani, C. Parker Gibbs. Forced induction of differentiation in osteosarcoma tumor initiating cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1188.

BMP Antagonist Gremlin 2 Limits Inflammation After Myocardial Infarction.

We have recently shown that the bone morphogenetic protein (BMP) antagonist Gremlin 2 (Grem2) is required for early cardiac development and cardiomyocyte differentiation. Our initial studies discovered that Grem2 is strongly induced in the adult heart after experimental myocardial infarction (MI). However, the function of Grem2 and BMP-signaling inhibitors after cardiac injury is currently unknown. To investigate the role of Grem2 during cardiac repair and assess its potential to improve ventricular function after injury. Our data show that Grem2 is transiently induced after MI in peri-infarct area cardiomyocytes during the inflammatory phase of cardiac tissue repair. By engineering loss- (Grem2(-/-)) and gain- (TG(Grem2)) of-Grem2-function mice, we discovered that Grem2 controls the magnitude of the inflammatory response and limits infiltration of inflammatory cells in peri-infarct ventricular tissue, improving cardiac function. Excessive inflammation in Grem2(-/-) mice after MI was because of overactivation of canonical BMP signaling, as proven by the rescue of the inflammatory phenotype through administration of the canonical BMP inhibitor, DMH1. Furthermore, intraperitoneal administration of Grem2 protein in wild-type mice was sufficient to reduce inflammation after MI. Cellular analyses showed that BMP2 acts with TNFα to induce expression of proinflammatory proteins in endothelial cells and promote adhesion of leukocytes, whereas Grem2 specifically inhibits the BMP2 effect. Our results indicate that Grem2 provides a molecular barrier that controls the magnitude and extent of inflammatory cell infiltration by suppressing canonical BMP signaling, thereby providing a novel mechanism for limiting the adverse effects of excessive inflammation after MI.

Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling.

Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF-1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF-1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF-1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)-mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF-1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1(Q207D/+) mouse model of FOP. Thus, an inflammation and cellular oxygen-sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF-1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIF-1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO. © 2016 American Society for Bone and Mineral Research.

A Survey of Strategies to Modulate the Bone Morphogenetic Protein Signaling Pathway: Current and Future Perspectives.

Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the TGF-β family of ligands and are unequivocally involved in regulating stem cell behavior. Appropriate regulation of canonical BMP signaling is critical for the development and homeostasis of numerous human organ systems, as aberrations in the BMP pathway or its regulation are increasingly associated with diverse human pathologies. In this review, we provide a wide-perspective on strategies that increase or decrease BMP signaling. We briefly outline the current FDA-approved approaches, highlight emerging next-generation technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways.

The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis.

Bone Morphogenetic Proteins (BMPs) form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

BMP/Smad signaling and embryonic cerebellum development: stem cell specification and heterogeneity of anterior rhombic lip.

The canonical bone morphogenetic proteins (BMPs) signaling have been shown to mediate many embryonic developmental processes. Due to its complexity, there are still many unknowns about this signal pathway including the Smad usage and requirement. Cerebellum, one of the most studied neural organs in development biology, requires canonical BMP signaling for stem cell specification. Here we review the role of canonical BMP signaling during the embryonic cerebellum development. Also, we raise several unsolved issues concerning the BMP signaling including the co-Smad independency of this signaling pathway. Besides, we also propose two models for explaining the cerebellar anterior rhombic lip (ARL) specification mechanisms. In addition, we review the heterogeneity of the ARL stem cells, which may provide new insight into understanding the neural stem cell specification process of the embryonic cerebellum.

An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.

CS-13 * BONE MORPHOGENETIC PROTEIN SIGNALING PROMOTES TUMORIGENESIS IN A TRANSGENIC MOUSE MODEL OF GLIOMA

Improved therapies for high grade glioma (HGG) are imperative, as the median survival after diagnosis with grade IV glioma is 15 months. Recently pathways regulating neural development, such as the bone morphogenetic protein (BMP) pathway, have been investigated as potential therapeutic targets in HGG. In xenograft transplant models, BMP signaling has been shown to have a tumor suppressive effect on the subpopulation of cells known as glioma-initiating or glioma stem cells. However, the degree to which BMP signaling plays a role in the bulk tumor cells or the more differentiated component in HGG is unknown. To investigate these questions we used both human samples of HGG and a novel murine model of human HGG established in our laboratory. We determined that BMP signaling is present and active in human HGG using a human HGG tissue-microarray. Our analysis of phospho-smad1/5/8 staining suggests that BMP signaling is present and active in most cells in many HGG gliomas, suggesting that BMP signaling is not limited to the glioma stem cell compartment. To examine the role of BMP signaling in differentiated, tumorigenic astrocytes, we deleted the BMP type IA receptor gene (Bmpr1a) in transformed astrocytes, effectively abrogating canonical BMP signaling in these cells. The cells were then transplanted orthotopically into immunocompetent adult host mice. Preliminary data suggest that, while BMP signaling may be tumor suppressive in stem-like cells, it acts as a tumor promoter in differentiated tumorigenic astrocytes. Compared to controls receiving cells with intact Bmpr1a, mice receiving Bmpr1a-knockout cells showed a significant increase in survival time upon orthotopic injection (21 vs. 52 days, p = 0.001). In vitro, deletion of Bmpr1a in oncogenic astrocytes resulted in decreased proliferation and increased Olig2 expression. Studies to further investigate the role of BMPs in HGG are underway.

C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation

The bone morphogenetic protein (BMP) signaling pathway regulates a wide range of cellular responses in metazoans. A key step in the canonical BMP signaling is the phosphorylation and activation of transcription factors Smad1, Smad5, and Smad8 (collectively Smad1/5/8) by the type I BMP receptors. We previously identified PPM1A as a phosphatase toward dephosphorylation of all receptor-regulated Smads (R-Smads), including Smad1/5/8. Here we report another nuclear phosphatase named SCP4/CTDSPL2, belonging to the FCP/SCP family, as a novel Smad phosphatase in the nucleus. SCP4 physically interacts with and specifically dephosphorylates Smad1/5/8, and as a result attenuates BMP-induced transcriptional responses. Knockdown of SCP4 in multipotent mesenchymal C2C12 cells leads to increased expression of BMP target genes and consequently promotes BMP-induced osteogenic differentiation. Collectively, our results demonstrate that SCP4, as a Smad phosphatase, plays a critical role in BMP-induced signaling and cellular functions.

Wnt7b Is an Important Intrinsic Regulator of Hair Follicle Stem Cell Homeostasis and Hair Follicle Cycling

The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis, and progeny differentiation. During morphogenesis, Wnt signaling is well-characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previously, we described the function of the bone morphogenetic protein (BMP) signaling target gene WNT7a in intrinsic regulation of hfSCs homeostasis in vivo. Here, we investigated the role of Wnt7b, which was also intrinsically upregulated in hfSCs during physiological and precocious anagen after BMP inhibition in vivo. We demonstrated Wnt7b to be a direct target of canonical BMP signaling in hfSCs and using Wnt7b conditional gene targeting during HF morphogenesis revealed disrupted HF cycling including a shorter anagen, premature catagen onset with overall shorter hair production, and diminished HF differentiation marker expression. Additionally, we observed that postnatal ablation of Wnt7b resulted in delayed HF activation, affecting both the hair germ and bulge hfSCs but still maintaining a two-step sequence of HF stimulation. Interestingly, Wnt7b cKO hfSCs participated in reformation of the new HF bulge, but with slower self-renewal. These findings demonstrate the importance of intrinsic Wnt7b expression in hfSCs regulation and normal HF cycling and surprisingly reveal a nonredundant role for Wnt7b in the control of HF anagen length and catagen entry which was not compensated by other Wnt ligands.

Smad1 and 5 but Not Smad8 Establish Stem Cell Quiescence Which Is Critical to Transform the Premature Hair Follicle During Morphogenesis Toward the Postnatal State

Hair follicles (HFs) are regenerative miniorgans that offer a highly informative model system to study the regulatory mechanisms of hair follicle stem cells (hfSCs) homeostasis and differentiation. Bone morphogenetic protein (BMP) signaling is key in both of these processes, governing hfSCs quiescence in the bulge and differentiation of matrix progenitors. However, whether canonical or noncanonical pathways of BMP signaling are responsible for these processes remains unresolved. Here, we conditionally ablated two canonical effectors of BMP signaling, Smad1 and Smad5 during hair morphogenesis and postnatal cycling in mouse skin. Deletion of Smad1 and Smad5 (dKO) in the epidermis during morphogenesis resulted in neonatal lethality with lack of visible whiskers. Interestingly, distinct patterns of phospho-Smads (pSmads) activation were detected with pSmad8 restricted to epidermis and pSmad1 and pSmad5 exclusively activated in HFs. Engraftment of dKO skin revealed retarded hair morphogenesis and failure to differentiate into visible hair. The formation of the prebulge and bulge reservoir for quiescent hfSCs was precluded in dKO HFs which remained in prolonged anagen. Surprisingly, in postnatal telogen HFs, pSmad8 expression was no longer limited to epidermis and was also present in dKO bulge hfSCs and matrix progenitors. Although pSmad8 activity alone could not prevent dKO hfSCs precocious anagen activation, it sustained efficient postnatal differentiation and regeneration of visible hairs. Together, our data suggest a pivotal role for canonical BMP signaling demonstrating distinguished nonoverlapping function of pSmad8 with pSmad1 and pSmad5 in hfSCs regulation and hair morphogenesis but a redundant role in adult hair progenitors differentiation.

Common Partner Smad-Independent Canonical Bone Morphogenetic Protein Signaling in the Specification Process of the Anterior Rhombic Lip during Cerebellum Development

Bone morphogenetic protein (BMP) signaling is critical for cerebellum development. However, the details of receptor regulated-Smad (R-Smad) and common partner Smad (Co-Smad, or Smad4) involvement are unclear. Here, we report that cerebellum-specific double conditional inactivation of Smad1 and Smad5 (Smad1/5) results in cerebellar hypoplasia, reduced granule cell numbers, and disorganized Purkinje neuron migration during embryonic development. However, single conditional inactivation of either Smad1 or Smad5 did not result in cerebellar abnormalities. Surprisingly, conditional inactivation of Smad4, which is considered to be the central mediator of canonical BMP-Smad signaling, resulted only in very mild cerebellar defects. Conditional inactivation of Smad1/5 led to developmental defects in the anterior rhombic lip (ARL), as shown by reduced cell proliferation and loss of Pax6 and Atoh1 expression. These defects subsequently caused the loss of the nuclear transitory zone and a region of the deep cerebellar nuclei. The normal maturation of the remaining granule cell precursors in the external granular layer (EGL) suggests Smad1/5 signaling is required for the specification process in ARL but not for the subsequent EGL development. Our results demonstrate functional redundancy for Smad1 and Smad5 but functional discrepancy between Smad1/5 and Smad4 during cerebellum development.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs).Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST.These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Sildenafil Potentiates Bone Morphogenetic Protein Signaling in Pulmonary Arterial Smooth Muscle Cells and in Experimental Pulmonary Hypertension

Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH), and BMPR-II deficiency contributes to idiopathic and experimental forms of PAH. Sildenafil, a potent type-5 nucleotide-dependent phosphodiesterase inhibitor, is an established treatment for PAH, but whether sildenafil affects bone morphogenetic protein (BMP) signaling in the pulmonary circulation remains unknown. Studies were undertaken in human pulmonary arterial smooth muscle cells (PASMCs) and in vivo in the monocrotaline rat model of PAH. In PASMCs, sildenafil enhanced BMP4-induced phosphorylation of Smad1/5, Smad nuclear localization, and Inhibitor of DNA binding protein 1 gene and protein expression. This effect was mimicked by 8-bromo-cyclic GMP. Pharmacological inhibition or small interfering RNA knockdown of cyclic GMP-dependent protein kinase I inhibited the effect of sildenafil on BMP signaling. In functional studies, we observed that sildenafil potentiated the antiproliferative effects of BMP4 on PASMC proliferation. Furthermore, sildenafil restored the antiproliferative response to BMP4 in PASMCs harboring mutations in BMPR-II. In the monocrotaline rat model of PAH, which is characterized by BMPR-II deficiency, sildenafil prevented the development of pulmonary hypertension and vascular remodeling, and partly restored Smad1/5 phosphorylation and Inhibitor of DNA binding protein 1 gene expression in vivo in monocrotaline exposed rat lungs. Sildenafil enhances canonical BMP signaling via cyclic GMP and cyclic GMP-dependent protein kinase I in vitro and in vivo, and partly restores deficient BMP signaling in BMPR-II mutant PASMCs. Our findings demonstrate a novel mechanism of action of sildenafil in the treatment of PAH and suggest that targeting BMP signaling may be beneficial in this disease.

Altered MicroRNA Processing in Heritable Pulmonary Arterial Hypertension

Heritable pulmonary arterial hypertension (HPAH) is primarily caused by mutations of the bone morphogenetic protein (BMP) type-II receptor (BMPR2). Recent identification of mutations in the downstream mediator Smad-8 (gene, SMAD9) was surprising, because loss of Smad-8 function in canonical BMP signaling is largely compensated by Smad-1 and -5. We therefore hypothesized that noncanonical pathways may play an important role in PAH. To determine whether HPAH mutations disrupt noncanonical Smad-mediated microRNA (miR) processing. Expression of miR-21, miR-27a, and miR-100 was studied in pulmonary artery endothelial (PAEC) and pulmonary artery smooth muscle cells (PASMC) from explant lungs of patients with PAH. SMAD9 mutation completely abrogated miR induction, whereas canonical signaling was only reduced by one-third. miR-21 levels actually decreased, suggesting that residual canonical signaling uses up or degrades existing miR-21. BMPR2 mutations also led to loss of miR induction in two of three cases. HPAH cells proliferated faster than other PAH or controls. miR-21 and miR-27a each showed antiproliferative effects in PAEC and PASMC, and PAEC growth rate after BMP treatment correlated strongly with miR-21 fold-change. Overexpression of SMAD9 corrected miR processing and reversed the hyperproliferative phenotype. HPAH-associated mutations engender a primary defect in noncanonical miR processing, whereas canonical BMP signaling is partially maintained. Smad-8 is essential for this miR pathway and its loss was not complemented by Smad-1 and -5; this may represent the first nonredundant role for Smad-8. Induction of miR-21 and miR-27a may be a critical component of BMP-induced growth suppression, loss of which likely contributes to vascular cell proliferation in HPAH.